Fig. 2. mDia1 regulates MyoD by an SRF-independent pathway. (A) Schematic of full-length mDia1 (FL) and five truncation derivatives. (B,C) mDia1N3 activates SRF. C2C12 myoblasts were co-transfected with full-length mDia1 (FL), mDia1 mutants or a GFP control, the SRF reporter and a -gal plasmid. To minimize effects of serum on SRF activity, transfected cells were incubated in 0.5% serum for 24 hours before assay. mDiaN3 increased SRF activity >25 fold (mean ± s.e.m., n=4, P<0.0041). Western blotting with anti-GFP (panel B, inset) showed that all mutants were expressed at relatively equal levels. (C) Quantification of MyoD expression detected by immunofluorescence assay in cells overexpressing GFP (control), full-length mDia1 (FL) or mDia1 mutants (N3, Hind3, F2, H+P, CC). Despite strongly activating SRF, mDia1N3 suppresses MyoD expression maximally (mean ± s.e.m., n=7, P<0.0001). (D) Immunodetection of MyoD expression in C2C12 myoblasts transiently transfected with GFP-tagged mDia1 truncation mutants (N3, H+P, CC).